Long PCR Is a Sensitive Method for Detecting Liberobacter asiaticum in Parasitoids Undergoing Risk Assessment in Quarantine

The accidental importation of plant pathogens in or on the bodies of parasitoids imported as natural enemies has been raised as a potential risk of classical biological control projects involving insects that serve as vectors of plant diseases. During quarantine evaluation of two parasitoids, Tamarixia radiata Waterston and Diaphorencyrtus aligarhensis (Shafee, Alam and Agarwal), imported for classical biological control of Asian citrus psylla Diaphorina citri (Kuwayama) in Florida, we were asked to determine whether these parasitoids were free of the causal agent of Asian greening disease, the bacterium Liberobacter asiaticum. Preliminary tests with allele-specific polymerase chain reaction (Standard PCR) suggested that the assays were prone to false negatives. Another PCR protocol, Long PCR, previously was shown to be more reliable than Standard PCR for screening of insects for another bacterium (Wolbachia). The sensitivity of Long and Standard PCR protocols was compared by use of plasmid DNA containing two DNA fragments from the greening disease agent or plasmid mixed with DNA extracted from host plants, psyllids, or parasitoids. Results indicated that inhibitors of the PCR were present in both plant and insect DNA, making the Standard PCR relatively insensitive and allowing high levels of false negatives. Long PCR, which incorporates a second DNA polymerase with proof-reading activity, yielded consistent results and was orders of magnitude more sensitive than the Standard PCR. As few as 100 copies of plasmid mixed with either plant or insect DNA consistently could be detected. Long PCR assays conducted on pooled and individual T. radiata and D. aligarhensis, their psyllid hosts, or their host plants over a period of 6 months failed to produce any positives, indicating that release of these two parasitoids should elicit little concern that greening bacteria would be introduced accidentally into Florida through this classical biological control program.